Project Summary The long-term goal of this project is to develop and translate into early phase clinical trials novel antibody-based reagents for the treatment of acute myelogenous leukemia (AML). Less than half of AML patients are cured with current treatment approaches, and relapse and refractory AML patients have a median overall survival of about 4 months. Allogeneic hematopoietic cell transplant (alloHCT) remains the only curative therapy for patients with relapsed and refractory AML. However, wider application of alloHCT has been limited by the morbidity associated with the procedure, which arises from graft-versus-host disease and from the toxicity of the conditioning regimen, a course of cytotoxic drugs and/or irradiation that is used to enable engraftment of donor hematopoietic stem and progenitor cells (HSPCs) in the bone marrow. In this proposal, we will utilize antibody-based immunotherapy approaches to treat AML relapse and allow for alloHCT without the need for chemotherapy and/or irradiation. In Aim 1, we will conduct a ?first-in-human? Phase I clinical trial of AMV564, a CD33 x CD3 tetravalent bispecific antibody in patients with relapsed or refractory AML (ClinicalTrials.gov id: NCT03144245). This study is designed in two segments: a Dose Escalation Stage testing up to 10 doses of AMV564 at 3 patients per dose, followed by an Expansion Stage segment at the recommended Phase 2 dose of AMV564. We will characterize the immunomodulatory activity and potential anti-tumor activity of AMV564 by completing correlative studies evaluating (1) serum cytokines, (2) AML and T cell subset numbers, phenotype and function and (3) the subclonal architecture of AML blasts. In Aim 2, we will determine if antibody-based drug conjugates can mitigate cytokine release syndrome (CRS) associated with T cell immunotherapeutics, kill AML blasts and provide conditioning for alloHSCT. T cell engaging therapies, which include bispecific retargeting reagents like AMV564, have been limited by CRS. We will test if targeted depletion of macrophage and monocyte populations with toxin-conjugated antibody fragments or modulation of the IFN? and IL-6 signaling pathways will prevent CRS mediated by T cell engaging therapies. If successful, these CRS-mitigating strategies would reduce the main limitation currently facing T cell engaging therapies. Next, we will test if toxin-conjugated antibody antigen binding fragments (Fabs) or bispecific antibodies targeting HSPCs/AML (via CD45 or c-Kit) and T lymphocytes/NK cells (via CD2 or CD7) can simultaneously treat AML and provide conditioning for alloHCT without the need for radiation or chemotherapy. The potential applications of this more targeted and less toxic conditioning regimen are not limited to alloHCT alone, but would also provide a platform for autologous gene therapy for inborn errors of metabolism, hemoglobinopathies, and primary immune deficiencies.